T Cells with Increased Immunosuppression Resistance

ABSTRACT

This invention relates to the treatment of cancer in an individual by administration of a population of modified T cells that express a recombinant cAMP phosphodiesterase (PDE) or a fragment thereof and an antigen receptor which binds specifically to cancer cells in the individual. Populations of modified T cells and methods of producing populations of modified T cells are provided, along with pharmaceutical compositions and methods of treatment

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119 or 365 to GBApplication No. 1616238.0, filed Sep. 23, 2016. The entire teachings ofthe above applications are incorporated herein by reference.

FIELD

The present invention relates to the modification of T cells to increasetheir resistance to immunosuppression and the use of modified T cells inimmunotherapy, for example for the treatment of cancer.

BACKGROUND

T cells (or T lymphocytes) are found widely distributed within tissuesand the tumour environment. T cells are distinguished from otherlymphocytes by the presence of T cell receptors (TCRs) on the cellsurface. The TCR is a multi-subunit transmembrane complex that mediatesthe antigen-specific activation of T cells. The TCR confers antigenspecificity on the T cell, by recognising an antigen peptide ligand thatis presented on the target cell by a major histocompatibility complex(MHC) molecule.

Although peptides derived from altered or mutated proteins in tumourtarget cells can be recognised as foreign by T cells expressing specificTCRs, many antigens on tumour cells are simply upregulated oroverexpressed (so called self-antigens) and do not induce a functional Tcell response. Therefore, studies have focussed on identifying targettumour antigens which are expressed, or highly expressed, in themalignant but not the normal cell type. Examples of such targets includethe cancer/testis (CT) antigen NY-ESO-1, which is expressed in a widearray of human cancers but shows restricted expression in normal tissues(Chen Y-T et al. Proc Natl Acad Sci USA. 1997; 94(5): 1914-1918), andthe MAGE-A family of CT antigens which are expressed in a very limitednumber of healthy tissues (Scanlan M. J. et al. Immunol Rev. 2002;188:22-32).

Identification of such antigens has promoted the development of targetedT cell-based immunotherapy, which has the potential to provide specificand effective cancer therapy (Ho, W. Y. et al. Cancer Cell 2003;3:1318-1328; Morris, E. C. et al. Clin. Exp. Immunol. 2003; 131:1-7;Rosenberg, S. A. Nature 2001; 411:380-384; Boon, T. and van der BruggenP. J. Exp. Med. 1996; 183:725-729).

However, the microenvironment of a tumour is often immunosuppressive andprevents the successful immunotherapy of cancer (Rabinovich G. A. et al.Annu Rev Immunol. 2007; 25:267-296). Extracellular adenosine is a knowninhibitor of immune function. High levels of adenosine in tumours havebeen found to play a significant role in the evasion of anti-tumourimmune responses (Blay J. et al. Cancer Res. 1997; 57:2602-2605; Ohta A.et al. Proc Natl Acad Sci USA. 2006; 103:13132-13137). Theadenosine-rich environment in tumours may induce T cell anergy (Zarek P.E. et al. Blood 2008; 111:251-259; Ohta A. et al. J Immunol. 2009;183:5487-5493), increase production of immunosuppressive cytokines(e.g., TGF-beta, IL-10) (Zarek P. E. et al. supra; Nowak M. et al. Eur JImmunol. 2010; 40:682-687), and discourage cellular immune responses bytargeting antigen-presenting cells (Hasko G. et al. Faseb J. 2000;14:2065-2074; Panther E. et al. Blood 2003; 101:3985-3990).

Prostaglandin E2 (PGE2) is also known to be an inhibitor of immunefunctions and has been widely demonstrated to suppress both innate andantigen-specific immunity (Phipps R. P. et al. Immunol Today. 1991;12:349-352; Harris S. G. et al. Trends Immunol. 2002; 23:144-150).

T cell-based immunotherapies which are more able to cope with thehostile tumour environment would be useful in providing more effectivecancer therapy.

SUMMARY

The present inventors have recognised that the cytotoxicity of T cellstargeting tumour cells may be increased by the inhibition of cAMPsignalling for example through recombinant expression of a cAMPphosphodiesterase (PDE) or fragment thereof. T cells modified to expressa cAMP PDE or fragment thereof may therefore be useful in cancerimmunotherapy.

An aspect of the invention provides a method of treating cancer in anindividual comprising;

-   -   administering to the individual a population of T cells that        express a recombinant cAMP phosphodiesterase (PDE) or a fragment        thereof and an antigen receptor which binds specifically to        cancer cells in the individual.

Another aspect of the invention provides a method of producing apopulation of modified T cells comprising;

-   -   providing a population of T cells obtained from an individual,        and    -   modifying the T cells to express a cAMP phosphodiesterase (PDE)        or a fragment thereof, thereby producing a population of        modified T cells.

In some embodiments, the T cells may express an endogenous antigenreceptor, such as a T cell receptor (TCR), which binds specifically tocancer cells from the individual. In other embodiments, a method mayfurther comprise modifying the T cells to express an antigen receptorwhich binds specifically to cancer cells from the individual. The Tcells may be modified to express the antigen receptor before, after orat the same time as they are modified to express the cAMP PDE orfragment.

Following modification, the population of modified T cells may beexpanded, stored and/or formulated into a pharmaceutical composition.

Another aspect of the invention provides a method of treating cancer inan individual comprising;

-   -   providing a population of T cells obtained from a donor        individual,    -   modifying the T cells to express a cAMP phosphodiesterase (PDE)        or a fragment thereof, thereby producing a population of        modified T cells, and    -   administering the population of modified T cells to a recipient        individual.

In some embodiments, the T cells may express an endogenous antigenreceptor which binds specifically to cancer cells from the donorindividual. In other embodiments, a method may further comprisemodifying the T cells to express an antigen receptor which bindsspecifically to cancer cells from the donor individual. The T cells maybe modified to express the antigen receptor before, after or at the sametime as they are modified to express the cAMP PDE or fragment.

The donor individual and the recipient individual may be the same (i.e.autologous treatment; the modified T cells are obtained from anindividual who is subsequently treated with the modified T cells) or thedonor individual and the recipient individual may be different (i.e.allogeneic treatment; the modified T cells are obtained from oneindividual and subsequently used to treat a different individual).

Another aspect of the invention provides a population of modified Tcells which express an antigen receptor which binds specifically tocancer cells and a cAMP phosphodiesterase (PDE) or fragment thereof,wherein said cells comprise a heterologous nucleic acid encoding thecAMP phosphodiesterase (PDE) or fragment thereof.

The antigen receptor may be endogenous or may be a recombinant antigenreceptor encoded by heterologous nucleic acid.

Other aspects of the invention provide pharmaceutical compositionscomprising the modified T cells described above, methods of treatmentcomprising administering the modified T cells or pharmaceuticalcompositions to an individual and modified T cells or pharmaceuticalcompositions for use in a method of treatment, for example, a method oftreatment of cancer in an individual.

Other aspects of the invention provide nucleic acid comprising anucleotide sequence encoding an antigen receptor which bindsspecifically to cancer cells and a nucleotide sequence encoding a cAMPphosphodiesterase (PDE) or fragment thereof, vectors comprising thenucleic acid, including lentiviral vectors, and methods of making viralparticles comprising the vectors.

BRIEF DESCRIPTION OF FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows plasmid maps of lentivectors expressing the NY-ESO^(c259) Tcell receptor in tandem with PDE4C (FIG. 1a ) or PDE7A (FIG. 1b ).

FIG. 2 shows target cell killing kinetics by NY-ESO specific T cellsthat overexpress PDE7A. FIG. 2a and FIG. 2b (data from two donors) showthat NY-ESO specific T cells overexpressing PDE7A have increased abilityto induce apoptosis in A375 melanoma target cells compared to NY-ESOspecific T cells alone.

FIG. 3 shows that overexpression of PDE7A in NY-ESO specific T cellsconfers resistance to inhibitory effects of prostaglandin E2. FIG. 3aand FIG. 3b (data from two donors) show that NY-ESO specific T cellsoverexpressing PDE7A are resistant to the T cell-inhibitory effects ofPGE2, demonstrating A375 melanoma target cell killing ability that iscomparable to that shown by the same T cells in the absence of PGE2.

FIG. 4 shows that overexpression of PDE7A in NY-ESO specific T cellsconfers partial resistance to the inhibitory effects of forskolin on Tcell killing ability. FIG. 4a and FIG. 4b (data from two donors) showthat NY-ESO specific T cells overexpressing PDE7A demonstrate superiorkilling ability in the presence of forskolin than NY-ESO specific Tcells that do not overexpress PDE7A (ADB869 T cells).

FIG. 5 shows that NY-ESO specific T cells overexpressing PDE7A arepartially resistant to the inhibition of cytokine release by mediatorsthat increase intracellular cAMP. Overexpression of PDE7A in NY-ESOspecific T cells partially overcomes the inhibitory effects of forskolin(FIG. 5a ), adenosine (FIG. 5b ) and PGE2 (FIG. 5c ) on IFN-γ secretion.

FIG. 6 shows that NY-ESO specific T cells overexpressing PDE4C arepartially resistant to the inhibition of cytokine release by mediatorsthat increase intracellular cAMP. FIG. 6a and FIG. 6b (data from twodonors) show that overexpression of PDE4C in NY-ESO specific T cellspartially blocks the inhibitory effects of PGE2 and forskolin on IFN-γsecretion.

FIG. 7 shows the results of a lentiviral transduction using the NY-ESOreceptor alone (NY-ESO1^(c259) TCR), NY-ESO receptor in combination withfull-length PDE7A (NY-ESO1^(c259) TCR+full length PDE7A) and NY-ESOreceptor in combination with truncated PDE7A (NY-ESO1^(c259)TCR+truncated PDE7A).

FIG. 8 shows the transduction efficiency and TCR expression levels forNY-ESO TCRs following transduction with full-length and truncated PDE7A.

FIG. 9 shows target cell killing kinetics by NY-ESO specific T cellsthat overexpress full-length or truncated PDE7A in the presence andabsence of PGE2. FIG. 9b shows comparable killing ability for allconstructs in the absence of inhibitors. FIG. 9c shows that NY-ESOspecific T cells overexpressing full-length or truncated PDE7Ademonstrate superior killing ability in the presence of PGE2 than NY-ESOspecific T cells that do not overexpress PDE7A.

FIG. 10 shows that NY-ESO specific T cells overexpressing full-length ortruncated PDE7A are partially resistant to the inhibition of cytokinerelease by mediators that increase intracellular cAMP. FIG. 10a and FIG.10b (data from two donors) show that overexpression of full-length ortruncated PDE7A in NY-ESO specific T cells partially blocks theinhibitory effects of PGE2 and forskolin on IFN-γ secretion.

DETAILED DESCRIPTION

This invention relates to methods of increasing the resistance ofanti-tumour T cells to the immunosuppressive microenvironment of tumoursthrough recombinant expression of a cAMP PDE or a fragment of a cAMPPDE. Reducing cAMP signalling through expression of a cAMP PDE orfragment thereof is shown herein to increase the resistance ofanti-tumour T cells to inhibition by adenosine and prostaglandin E2(PGE2). Modified T cells with increased resistance to inhibition maydisplay increased cytotoxicity and/or cytokine release relative tounmodified T cells. Anti-tumour T cells modified to express cAMP PDE ora cAMP PDE fragment as described herein may be useful in immunotherapy,for example adoptive cell transfer (ACT) for the treatment of cancer.

T cells (also called T lymphocytes) are white blood cells that play acentral role in cell-mediated immunity. T cells can be distinguishedfrom other lymphocytes by the presence of a T cell receptor (TCR) on thecell surface. There are several types of T cells, each type having adistinct function.

T helper cells (T_(H) cells) are known as CD4⁻ T cells because theyexpress the CD4 surface glycoprotein. CD4⁺ T cells play an importantrole in the adaptive immune system and help the activity of other immunecells by releasing T cell cytokines and helping to suppress or regulateimmune responses. They are essential for the activation and growth ofcytotoxic T cells.

Cytotoxic T cells (T_(C) cells, CTLs, killer T cells) are known as CD8⁺T cells because they express the CD8 surface glycoprotein. CD8⁺ T cellsact to destroy virus-infected cells and tumour cells. Most CD8⁺ T cellsexpress TCRs that can recognise a specific antigen displayed on thesurface of infected or damaged cells by a class I MHC molecule. Specificbinding of the TCR and CD8 glycoprotein to the antigen and MHC moleculeleads to T cell-mediated destruction of the infected or damaged cells.

T cells for use as described herein may be CD4⁺ T cells; CD8⁻ T cells;or CD4⁺ T cells and CD8⁺ T cells. For example, the T cells may be amixed population of CD4⁺ T cells and CD8⁺ T cells.

Suitable T cells for use as described herein may be obtained from adonor individual. In some embodiments, the donor individual may be thesame person as the recipient individual to whom the T cells will beadministered following modification and expansion as described herein(autologous treatment). In other embodiments, the donor individual maybe a different person to the recipient individual to whom the T cellswill be administered following modification and expansion as describedherein (allogeneic treatment). For example, the donor individual may bea healthy individual who is human leukocyte antigen (HLA) matched(either before or after donation) with a recipient individual sufferingfrom cancer.

A method described herein may comprise the step of obtaining T cellsfrom an individual and/or isolating T cells from a sample obtained froman individual with cancer.

A population of T cells may be isolated from a blood sample. Suitablemethods for the isolation of T cells are well known in the art andinclude, for example fluorescent activated cell sorting (FACS: see forexample, Rheinherz et al (1979) PNAS 76 4061), cell panning (see forexample, Lum et al (1982) Cell Immunol 72 122) and isolation usingantibody coated magnetic beads (see, for example, Gaudernack et al 1986J Immunol Methods 90 179).

CD4⁺ and CD8⁺ T cells may be isolated from the population of peripheralblood mononuclear cells (PBMCs) obtained from a blood sample. PBMCs maybe extracted from a blood sample using standard techniques. For example,ficoll may be used in combination with gradient centrifugation (Böyum A.Scand J Clin Lab Invest. 1968; 21(Suppl0.97):77-89), to separate wholeblood into a top layer of plasma, followed by a layer of PBMCs and abottom fraction of polymorphonuclear cells and erythrocytes. In someembodiments, the PBMCs may be depleted of CD14⁺ cells (monocytes).

Following isolation, the T cells may be activated. Suitable methods foractivating T cells are well known in the art. For example, the isolatedT cells may be exposed to a T cell receptor (TCR) agonist. Suitable TCRagonists include ligands, such as a peptide displayed on a class I or IIMHC molecule on the surface of an antigen presenting cell, such as adendritic cell, and soluble factors, such as anti-TCR antibodies.

An anti-TCR antibody may specifically bind to a component of the TCR,such as εCD3, αCD3 or αCD28. Anti-TCR antibodies suitable for TCRstimulation are well-known in the art (e.g. OKT3) and available fromcommercial suppliers (e.g. eBioscience CO USA). In some embodiments, Tcells may be activated by exposure to anti-αCD3 antibodies and IL2. Morepreferably, T cells are activated by exposure to anti-αCD3 antibodiesand anti-αCD28 antibodies. The activation may occur in the presence orabsence of CD14⁻ monocytes. Preferably, the T cells may be activatedwith anti-CD3 and anti-CD28 antibody coated beads. For example, PBMCs orT cell subsets including CD4⁺ and/or CD8⁺ cells may be activated,without feeder cells (antigen presenting cells) or antigen, usingantibody coated beads, for example magnetic beads coated with anti-CD3and anti-CD28 antibodies, such as Dynabeads® Human T-Activator CD3/CD28(ThermoFisher Scientific).

Following isolation and activation, the T cells may be modified toexpress cyclic adenosine monophosphate (cAMP) phosphodiesterase (PDE) ora fragment of a cAMP PDE.

The cAMP PDE or fragment inhibits cAMP signalling in a cell byhydrolysing phosphodiester bonds and catalysing the decomposition ofadenosine 3′, 5′-cyclic phosphate (cyclic adenosine monophosphate; cAMP)to adenosine 5′-phosphate (adenosine monophosphate; AMP) (EC3.1.4.17; EC3.1.4.53) and/or by directly inhibiting cAMP dependent protein kinase(PKA) (see for example, Han et al (2006) JBC 281 22 15050-15057). Forexample, a cAMP PDE or cAMP PDE fragment may (i) inhibit the catalytic(C) subunit of cAMP dependent protein kinase (PKA) (ii) catalyse thedecomposition of cAMP, or (iii) both (i) and (ii).

Suitable cAMP PDEs are well-known in the art and include PDE1, PDE2,PDE3, PDE4, PDE7, PDE8, PDE10 and PDE11. In some preferred embodiments,the cAMP PDE may be PDE4 or PDE7.

PDE7 may include PDE7A (Gene ID 5150) and may comprise the amino acidsequence having the database reference CCDS56538.1; NP_001229247.1 GI:334085277 (SEQ ID NO: 1) or CCDS34901.1; NP_002594.1 GI: 24429566.

PDE4 may include PDE4A (Gene ID 5141) and PDE4C (Gene ID 5143). PDE4Amay comprise the amino acid sequence having the database referenceCCDS45961.1; NP_001104777.1 GI: 162329608 (SEQ ID NO: 2) and PDE4C maycomprise the amino acid sequence having the database referenceCCDS12373.1; NP_000914.2 GI: 115529445 (SEQ ID NO: 3).

The amino acid sequences of other cAMP PDEs are well known in the artand available on public databases.

A cAMP PDE may comprise a cAMP PDE reference amino acid sequence citedherein or may be a variant thereof. A variant may have an amino acidsequence having at least 20%, at least 25%, at least 30%, at least 40%,at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98% or atleast 99% sequence identity to the reference amino acid sequence.

Amino acid sequence identity is generally defined with reference to thealgorithm GAP (GCG Wisconsin Package™, Accelrys, San Diego Calif.). GAPuses the Needleman & Wunsch algorithm (J. Mol. Biol. (48): 444-453(1970)) to align two complete sequences that maximizes the number ofmatches and minimizes the number of gaps. Generally, the defaultparameters are used, with a gap creation penalty=12 and gap extensionpenalty=4. Use of GAP may be preferred but other algorithms may be used,e.g. BLAST, psiBLAST or TBLASTN (which use the method of Altschul et al.(1990) J Mol. Biol. 215: 405-410), FASTA (which uses the method ofPearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Watermanalgorithm (Smith and Waterman (1981) J Mol Biol. 147: 195-197),generally employing default parameters.

Particular amino acid sequence variants may differ from a referencesequence by insertion, addition, substitution or deletion of 1 aminoacid, 2, 3, 4, 5-10, 10-20 or 20-30 amino acids. In some embodiments, avariant sequence may comprise the reference sequence with 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more residues inserted, deleted or substituted. Forexample, up to 15, up to 20, up to 30 or up to 40 residues may beinserted, deleted or substituted.

In some preferred embodiments, a variant may differ from a referencesequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservativesubstitutions. Conservative substitutions involve the replacement of anamino acid with a different amino acid having similar properties. Forexample, an aliphatic residue may be replaced by another aliphaticresidue, a non-polar residue may be replaced by another non-polarresidue, an acidic residue may be replaced by another acidic residue, abasic residue may be replaced by another basic residue, a polar residuemay be replaced by another polar residue or an aromatic residue may bereplaced by another aromatic residue. Conservative substitutions may,for example, be between amino acids within the following groups:

-   -   (i) alanine and glycine;    -   (ii) glutamic acid, aspartic acid, glutamine, and asparagine    -   (iii) arginine and lysine;    -   (iv) asparagine, glutamine, glutamic acid and aspartic acid    -   (v) isoleucine, leucine and valine;    -   (vi) phenylalanine, tyrosine and tryptophan    -   (vii) serine, threonine, and cysteine.

A fragment of a cAMP PDE is a truncated sequence which contains lessthan the full-length cAMP PDE sequence but which retains some or all ofthe cAMP signalling inhibition activity. A fragment may be a catalyticfragment that displays cAMP PDE activity or, more preferably anon-catalytic fragment, for example a fragment that inhibits cAMPdependent protein kinase (PKA). A fragment of a full-length cAMP PDEsequence may comprise at least 40 amino acids, at least 50 amino acidsor at least 60 contiguous amino acids from the full-length cAMP PDEsequence. A fragment may comprise 60 or fewer, 100 or fewer, 200 orfewer or 300 or fewer amino acid residues.

In some embodiments, a cAMP PDE may bind and inhibit the catalytic (C)subunit of cAMP dependent protein kinase (PKA). A suitable fragment maycomprise one or more copies of a 16-22 amino acid repeat sequence,preferably about 18 amino acids, comprising a PKA pseudosubstrate site,for example a RRGAI motif. A suitable repeat sequence may have the aminoacid sequence;

-   -   P (V/N) P (Q/R) (H/Q) (V/L) (L/S) (S/Q) RRGAIS (F/Y) (S/S) SS

Examples of repeat sequences are highlighted below in SEQ ID NO: 4. Afragment of a full-length cAMP PDE sequence may be an N terminalfragment i.e. the N terminal residue of the fragment may correspond tothe N terminal residue of full-length cAMP PDE sequence. For example, anN terminal fragment may comprise at least 40, at least 50, at least 60,at least 75 or at least 100 contiguous amino acids from the N terminalof the full-length cAMP PDE sequence. In some embodiments, a suitable Nterminal fragment may comprise amino acids 1 to 57 of SEQ ID NO: 4. Inother embodiments, a suitable N terminal fragment may comprise SEQ IDNO: 4.

The recombinant cAMP PDE or fragments thereof expressed in the T cellsmay comprise a heterologous tag at the C terminal or more preferably theN terminal. A tag is a peptide sequence which is not naturallyassociated with the cAMP PDE and which forms one member of a specificbinding pair. T cells that express the recombinant cAMP PDE may beidentified and/or purified by the binding of the other member of thespecific binding pair to the tag. For example, the tag may form anepitope which is bound by an anti-tag antibody. This may be useful inidentifying modified T cells during treatment.

Suitable tags include for example, MRGS(H)₆, DYKDDDDK (FLAG™), T7-,S-(KETAAAKFERQHMDS), poly-Arg (R₅₋₆), poly-His (H₂₋₁₀), poly-Cys (C₄)poly-Phe(F₁₁) poly-Asp(D₅₋₁₆), SUMO tag (Invitrogen Champion pET SUMOexpression system), Strept-tag II (WSHPQFEK), c-myc (EQKLISEEDL),Influenza-HA tag (Murray, P. J. et al (1995) Anal Biochem 229, 170-9),Glu-Glu-Phe tag (Stammers, D. K. et al (1991) FEBS Len 283, 298-302),Tag.100 (Qiagen; 12 aa tag derived from mammalian MAP kinase 2), Cruztag 09™ (MKAEFRRQESDR, Santa Cruz Biotechnology Inc.) and Cruz tag 22™(MRDALDRLDRLA, Santa Cruz Biotechnology Inc.). Known tag sequences arereviewed in Terpe (2003) Appl. Microbiol. Biotechnol. 60 523-533. Inpreferred embodiments, a haemagglutinin (HA) tag, such as YPYDVPDYA, maybe used.

The cAMP PDE or fragment expressed in the modified T cell is arecombinant protein that is encoded by a heterologous nucleic acid i.e.the cAMP PDE is expressed from encoding nucleic acid that has beenincorporated into the genome of the T cell by recombinant techniques.cAMP signalling in the modified T cells may be reduced relative to cAMPsignalling in unmodified T cells. In some embodiments, cAMP PDE activityin the modified T cells may be at least 5 fold higher, at least 10 foldhigher or at least 100 fold higher that cAMP PDE activity in unmodifiedT cells.

Modification of a T cell to express the cAMP PDE or fragment maycomprise introducing the heterologous nucleic acid encoding the cAMP PDEor fragment into the T cell. Suitable methods for the introduction andexpression of heterologous nucleic acids into T cells are well-known inthe art and described in more detail below.

Following introduction, a modified T cell as described herein maycomprise one or more than one copy of the heterologous nucleic acidencoding the cAMP PDE or fragment.

A modified T cell as described herein also expresses an antigen receptorthat binds specifically to cancer cells.

The antigen receptor may be a T cell receptor (TCR). TCRs aredisulphide-linked membrane anchored heterodimeric proteins, typicallycomprising highly variable alpha (α) and beta (β) chains expressed as acomplex with invariant CD3 chain molecules. T cells expressing thesetype of TCRs are referred to as α:β (or αβ) T cells. A minority of Tcells express an alternative TCR comprising variable gamma (γ) and delta(δ) chains and are referred to as γδ T cells.

Suitable TCRs bind specifically to a major histocompatibility complex(MHC) on the surface of cancer cells that displays a peptide fragment ofa tumour antigen. An MHC is a set of cell-surface proteins which allowthe acquired immune system to recognise ‘foreign’ molecules. Proteinsare intracellularly degraded and presented on the surface of cells bythe MHC. MHCs displaying ‘foreign’ peptides, such a viral or cancerassociated peptides, are recognised by T cells with the appropriateTCRs, prompting cell destruction pathways. MHCs on the surface of cancercells may display peptide fragments of tumour antigen i.e. an antigenwhich is present on a cancer cell but not the correspondingnon-cancerous cell. T cells which recognise these peptide fragments mayexert a cytotoxic effect on the cancer cell.

In some embodiments, the TCR that binds specifically to cancer cells maybe naturally expressed by the T cells (i.e. an endogenous TCR). Forexample, the T cells may be Tumour Infiltrating Lymphocytes (TILs). TILsmay be obtained from an individual with a cancer condition usingstandard techniques.

More preferably, the TCR is not naturally expressed by the T cells (i.e.the TCR is exogenous or heterologous). Heterologous TCRs may includeαβTCR heterodimers. Suitable heterologous TCRs may bind specifically tocancer cells that express a tumour antigen. For example, the T cells maybe modified to express a heterologous TCR that binds specifically toMHCs displaying peptide fragments of a tumour antigen expressed by thecancer cells in a specific cancer patient. Tumour antigens expressed bycancer cells in the cancer patient may identified using standardtechniques.

A heterologous TCR may be a synthetic or artificial TCR i.e. a TCR thatdoes not exist in nature. For example, a heterologous TCR may beengineered to increase its affinity or avidity for a tumour antigen(i.e. an affinity enhanced TCR). The affinity enhanced TCR may compriseone or more mutations relative to a naturally occurring TCR, forexample, one or more mutations in the hypervariable complementaritydetermining regions (CDRs) of the variable regions of the TCR α and βchains. These mutations increase the affinity of the TCR for MHCs thatdisplay a peptide fragment of a tumour antigen expressed by cancercells. Suitable methods of generated affinity enhanced TCRs includescreening libraries of TCR mutants using phage or yeast display and arewell known in the art (see for example Robbins et al J Immunol (2008)180(9):6116; San Miguel et al (2015) Cancer Cell 28 (3) 281-283; Schmittet al (2013) Blood 122 348-256; Jiang et al (2015) Cancer Discovery 5901).

Preferred affinity enhanced TCRs may bind to cancer cells expressing oneor more of the tumour antigens NY-ESO1, PRAME, alpha-fetoprotein (AFP),MAGE A4, MAGE A1, MAGE A10 and MAGE B2.

Alternatively, the antigen receptor may be a chimeric antigen receptor(CAR). CARs are artificial receptors that are engineered to contain animmunoglobulin antigen binding domain, such as a single-chain variablefragment (scFv). A CAR may, for example, comprise an scFv fused to a TCRCD3 transmembrane region and endodomain. An scFv is a fusion protein ofthe variable regions of the heavy (V_(H)) and light (V_(L)) chains ofimmunoglobulins, which may be connected with a short linker peptide ofapproximately 10 to 25 amino acids (Huston J. S. et al. Proc Natl AcadSci USA 1988; 85(16):5879-5883). The linker may be glycine-rich forflexibility, and serine or threonine rich for solubility, and mayconnect the N-terminus of the V_(H) to the C-terminus of the V_(L), orvice versa. The scFv may be preceded by a signal peptide to direct theprotein to the endoplasmic reticulum, and subsequently the T cellsurface. In the CAR, the scFv may be fused to a TCR transmembrane andendodomain. A flexible spacer may be included between the scFv and theTCR transmembrane domain to allow for variable orientation and antigenbinding. The endodomain is the functional signal-transmitting domain ofthe receptor. An endodomain of a CAR may comprise, for example,intracellular signalling domains from the CD3ζ-chain, or from receptorssuch as CD28, 41BB, or ICOS. A CAR may comprise multiple signallingdomains, for example, but not limited to, CD3z-CD28-41BB orCD3z-CD28-OX40.

The CAR may bind specifically to a tumour-specific antigen expressed bycancer cells. For example, the T cells may be modified to express a CARthat binds specifically to a tumour antigen that is expressed by thecancer cells in a specific cancer patient. Tumour antigens expressed bycancer cells in the cancer patient may identified using standardtechniques.

Expression of a heterologous antigen receptor, such as a heterologousTCR or CAR, may alter the immunogenic specificity of the T cells so thatthey recognise or display improved recognition for one or more tumourantigens that are present on the surface of the cancer cells of anindividual with cancer.

In some embodiments, the T cells may display reduced binding or nobinding to cancer cells in the absence of the heterologous antigenreceptor. For example, expression of the heterologous antigen receptormay increase the affinity and/or specificity of the cancer cell bindingof modified T cells relative to unmodified T cells.

The term “heterologous” refers to a polypeptide or nucleic acid that isforeign to a particular biological system, such as a host cell, and isnot naturally present in that system. A heterologous polypeptide ornucleic acid may be introduced to a biological system by artificialmeans, for example using recombinant techniques. For example,heterologous nucleic acid encoding a polypeptide may be inserted into asuitable expression construct which is in turn used to transform a hostcell to produce the polypeptide. A heterologous polypeptide or nucleicacid may be synthetic or artificial or may exist in a differentbiological system, such as a different species or cell type. Anendogenous polypeptide or nucleic acid is native to a particularbiological system, such as a host cell, and is naturally present in thatsystem. A recombinant polypeptide is expressed from heterologous nucleicacid that has been introduced into a cell by artificial means, forexample using recombinant techniques. A recombinant polypeptide may beidentical to a polypeptide that is naturally present in the cell or maybe different from the polypeptides that are naturally present in thatcell.

T cells may be modified to express a heterologous antigen receptor whichspecifically binds to the cancer cells of a cancer patient. The cancerpatient may be subsequently treated with the modified T cells. Suitablecancer patients for treatment with the modified T cells may beidentified by a method comprising;

-   -   obtaining sample of cancer cells from an individual with cancer        and;    -   identifying the cancer cells as binding to the antigen receptor        expressed by the modified T cells.

Cancer cells may be identified as binding to the antigen receptor byidentifying one or more tumour antigens expressed by the cancer cells.Methods of identifying antigens on the surface of cancer cells obtainedfrom an individual with cancer are well-known in the art.

In some embodiments, a heterologous antigen receptor suitable for thetreatment of a specific cancer patient may be identified by;

-   -   obtaining sample of cancer cells from an individual with cancer        and;    -   identifying an antigen receptor that specifically binds to the        cancer cells.

An antigen receptor that specifically binds to the cancer cells may beidentified for example by identifying one or more tumour antigensexpressed by the cancer cells. Methods of identifying antigens on thesurface of cancer cells obtained from an individual with cancer arewell-known in the art. An antigen receptor which binds to the one ormore tumour antigens or which binds to MHC-displayed peptide fragmentsof the one or more antigens may then be identified, for example fromantigen receptors of known specificities or by screening a panel orlibrary of antigen receptors with diverse specificities. Antigenreceptors that specifically bind to cancer cells having one or moredefined tumour antigens may be produced using routine techniques.

The T cells may be modified to express the identified antigen receptoras described herein.

The cancer cells of an individual suitable for treatment as describedherein may express the antigen and may be of correct HLA type to bindthe antigen receptor.

Cancer cells may be distinguished from normal somatic cells in anindividual by the expression of one or more antigens (i.e. tumourantigens). Normal somatic cells in an individual may not express the oneor more antigens or may express them in a different manner, for exampleat lower levels, in different tissue and/or at a different developmentalstage. Tumour antigens may elicit immune responses in the individual. Inparticular, a tumour antigen may elicit a T cell-mediated immuneresponse against cancer cells in the individual that express the tumourantigen. One or more tumour antigens expressed by cancer cells in apatient may be selected as a target antigen for heterologous receptorson modified T cells.

Tumour antigens expressed by cancer cells may include, for example,cancer-testis (CT) antigens encoded by cancer-germ line genes, such asMAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8,MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, GAGE-I, GAGE-2, GAGE-3, GAGE-4,GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-I, RAGE-1, LB33/MUM-1, PRAIVIE,NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4),MAGE-C1/CT7, MAGE-C2, NY-ESO-I, LAGE-I, SSX-I, SSX-2(HOM-MEL-40), SSX-3,SSX-4, SSX-5, SCP-I and XAGE and immunogenic fragments thereof (Simpsonet al. Nature Rev (2005) 5, 615-625, Gure et al., Clin Cancer Res (2005)11, 8055-8062; Velazquez et al., Cancer Immun (2007) 7, 11 ; Andrade etal., Cancer Immun (2008) 8, 2; Tinguely et al., Cancer Science (2008);Napoletano et al., Am J of Obstet Gyn (2008) 198, 99 e91-97).

Other tumour antigens include, for example, overexpressed, upregulatedor mutated proteins and differentiation antigens particularly melanocytedifferentiation antigens such as p53, ras, CEA, MUC1, PMSA, PSA,tyrosinase, Melan-A, MART-1, gp100, gp75, alpha-actinin-4, Bcr-Ablfusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1,dek-can fusion protein, EF2, ETV6-AML1 fusion protein,LDLR-fucosyltransferaseAS fusion protein, HLA-A2, HLA-A11, hsp70-2,KIAAO205, Mart2, Mum-2, and 3, neo-PAP, myosin class I, OS-9,pml-RAR.alpha. fusion protein, PTPRK, K-ras, N-ras, triosephosphateisomerase, GnTV, Herv-K-mel, NA-88, SP17, and TRP2-Int2, (MART-I),E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA,human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5,MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9,CA 72-4, CAM 17.1, NuMa, K-ras, alpha.-fetoprotein, 13HCG, BCA225, BTAA,CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1,CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag,MOV18, NB\170K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 bindingprotein\cyclophilin C-associated protein), TAAL6, TAG72, TLP, TPS andtyrosinase related proteins such as TRP-1, TRP-2.

Other tumour antigens include out-of-frame peptide-MHC complexesgenerated by the non-AUG translation initiation mechanisms employed by“stressed” cancer cells (Malarkannan et al. Immunity 1999 Jun;10(6):681-90).

Other tumour antigens are well-known in the art (see for exampleWO00/20581; Cancer Vaccines and Immunotherapy (2000) Eds Stern, Beverleyand Carroll, Cambridge University Press, Cambridge) The sequences ofthese tumour antigens are readily available from public databases butare also found in WO 1992/020356 A1, WO 1994/005304 A1, WO 1994/023031A1, WO 1995/020974 A1, WO 1995/023874 A1 and WO 1996/026214 A1.

Preferred tumour antigens include NY-ESO1, PRAME, alpha-fetoprotein(AFP), MAGE A4, MAGE A1, MAGE A10 and MAGE B2, most preferably NY-ESO-1and MAGE-A10.

NY-ESO-1 is a human tumour antigen of the cancer/testis (CT) family andis frequently expressed in a wide variety of cancers, includingmelanoma, prostate, transitional cell bladder, breast, lung, thyroid,gastric, head and neck, and cervical carcinoma (van Rhee F. et al. Blood2005; 105(10): 3939-3944). In addition, expression of NY-ESO-1 isusually limited to germ cells and is not expressed in somatic cells(Scanlan M. J. et al. Cancer Immun. 2004; 4(1)). Suitable affinityenhanced TCRs that bind to cancer cells expressing NY-ESO-1 includeNY-ESO-1^(c) ²⁵⁹ .

NY-ESO-1 c²⁵⁹ is an affinity enhanced TCR is mutated at positions 95 and96 of the alpha chain 95:96LY relative to the wildtype TCR. NY-ESO-1c²⁵⁹ binds to a peptide corresponding to amino acid residues 157-165 ofthe human cancer testis Ag NY-ESO-1 (SLLMWITQC) in the context of theHLA-A2+ class 1 allele with increased affinity relative to theunmodified wild type TCR (Robbins et al J Immunol (2008) 180(9):6116).

MAGE-A10 is a highly immunogenic member of the MAGE-A family of CTantigens, and is expressed in germ cells but not in healthy tissue.MAGE-A10 is expressed in high percentages of cancer cells from a numberof tumours (Schultz-Thater E. et al. Int J Cancer. 2011;129(5):1137-1148).

The modification of T cells and their subsequent expansion may beperformed in vitro and/or ex vivo.

T cells may be modified to express a cAMP PDE or fragment of a cAMP PDE,and, optionally an antigen receptor, by the introduction of heterologousencoding nucleic acid into the T cells.

In some embodiments, heterologous nucleic acid encoding cAMP PDE orfragment of a cAMP PDE and antigen receptor are introduced into the Tcells in the same expression vector. This may be helpful in increasingthe proportion of T cells which express both genes after transduction.In other embodiments, heterologous nucleic acid encoding cAMP PDE andantigen receptor may be introduced into the T cells in differentexpression vectors.

The cAMP PDE or fragment and the antigen receptor may be expressed inthe same transcript as a fusion protein and subsequently separated, forexample using a site-specific protease. Alternatively, the cAMP PDE orfragment and the antigen receptor may be expressed in differenttranscripts.

For example, a fusion protein comprising truncated cAMP PDE and anNY-ESO TCR may be expressed. The fusion protein may comprise the aminoacid sequence of SEQ ID NO: 6 or a variant thereof and may be encoded bya nucleic acid having the nucleotide sequence of SEQ ID NO: 5 or avariant thereof. Alternatively, a fusion protein comprising truncatedcAMP PDE and an MAGE-A4 TCR may be expressed. The fusion protein maycomprise the amino acid sequence of SEQ ID NO: 8 or a variant thereofand may be encoded by a nucleic acid having the nucleotide sequence ofSEQ ID NO: 7 or a variant thereof.

Nucleic acid encoding an antigen receptor may encode all the sub-unitsof the receptor. For example, nucleic acid encoding a TCR may comprise anucleotide sequence encoding a TCR α chain and a nucleotide sequenceencoding a TCR β chain.

Nucleic acid may be introduced into the T cells by any convenientmethod. When introducing or incorporating a heterologous nucleic acidinto a T cell, certain considerations must be taken into account,well-known to those skilled in the art. The nucleic acid to be insertedshould be assembled within a construct or vector which containseffective regulatory elements which will drive transcription in the Tcell. Suitable techniques for transporting the constructor vector intothe T cell are well known in the art and include calcium phosphatetransfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.vaccinia or lentivirus. For example, solid-phase transduction may beperformed without selection by culture on retronectin-coated, retroviralvector-preloaded tissue culture plates.

Preferably, nucleic acid encoding a cAMP PDE or fragment and,optionally, an antigen receptor may be contained in a viral vector, mostpreferably a gamma retroviral vector or a lentiviral vector, such as aVSVg-pseudotyped lentiviral vector. The T cells may be transduced bycontact with a viral particle comprising the nucleic acid. Viralparticles for transduction may be produced according to known methods.For example, HEK293T cells may be transfected with plasmids encodingviral packaging and envelope elements as well as a lentiviral vectorcomprising the coding nucleic acid. A VSVg-pseudotyped viral vector maybe produced in combination with the viral envelope glycoprotein G of theVesicular stomatitis virus (VSVg) to produce a pseudotyped virusparticle.

Many known techniques and protocols for manipulation and transformationof nucleic acid, for example in preparation of nucleic acid constructs,introduction of DNA into cells and gene expression are described indetail in Protocols in Molecular Biology, Second Edition, Ausubel et al.eds. John Wiley & Sons, 1992.

Following the introduction of nucleic acid into the T cells, the initialpopulation of modified T cells may be cultured in vitro such that themodified T cells proliferate and expand the population.

The modified T cell population may for example be expanded usingmagnetic beads coated with anti-CD3 and anti-CD28. The modified T cellsmay be cultured using any convenient technique to produce the expandedpopulation. Suitable culture systems include stirred tank fermenters,airlift fermenters, roller bottles, culture bags or dishes, and otherbioreactors, in particular hollow fibre bioreactors. The use of suchsystems is well-known in the art.

Numerous culture media suitable for use in the proliferation of T cellsex vivo are available, in particular complete media, such as AIM-V,Iscoves medium and RPMI-1640 (Invitrogen-GIBCO). The medium may besupplemented with other factors such as serum, serum proteins andselective agents. For example, in some embodiments, RPMI-1640 mediumcontaining 2 mM glutamine, 10% FBS, 25 mM HEPES, pH 7.2, 1%penicillin-streptomycin, and 55 μM β-mercaptoethanol and optionallysupplemented with 20 ng/ml recombinant IL-2 may be employed. The culturemedium may be supplemented with the agonistic or antagonist factorsdescribed above at standard concentrations which may readily bedetermined by the skilled person by routine experimentation.

Conveniently, cells are cultured at 37° C. in a humidified atmospherecontaining 5% CO₂ in a suitable culture medium.

Methods and techniques for the culture of T cells and other mammaliancells are well-known in the art (see, for example, Basic Cell CultureProtocols, C. Helgason, Humana Press Inc. U.S. (15 Oct. 2004) ISBN:1588295451; Human Cell Culture Protocols (Methods in Molecular MedicineS.) Humana Press Inc., U.S. (9 Dec. 2004) ISBN: 1588292223; Culture ofAnimal Cells: A Manual of Basic Technique, R. Freshney, John Wiley &Sons Inc (2 Aug. 2005) ISBN: 0471453293, Ho WY et al J Immunol Methods.(2006) 310:40-52)

In some embodiments, it may be convenient to isolate and/or purify themodified T cells from the population. Any convenient technique may beused, including FACS and antibody coated magnetic particles.

Optionally, the population of modified T cells produced as describedherein may be stored, for example by lyophilisation and/orcryopreservation, before use.

A population of modified T cells may be admixed with other reagents,such as buffers, carriers, diluents, preservatives and/orpharmaceutically acceptable excipients. Suitable reagents are describedin more detail below. A method described herein may comprise admixingthe population of modified T cells with a pharmaceutically acceptableexcipient.

Pharmaceutical compositions suitable for administration (e.g. byinfusion), include aqueous and non-aqueous isotonic, pyrogen-free,sterile injection solutions which may contain anti-oxidants, buffers,preservatives, stabilisers, bacteriostats, and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. Examples of suitable isotonic vehicles foruse in such formulations include Sodium Chloride Injection, Ringer'sSolution, or Lactated Ringer's Injection. Suitable vehicles can be foundin standard pharmaceutical texts, for example, Remington'sPharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton,Pa., 1990.

In some preferred embodiments, the modified T cells may be formulatedinto a pharmaceutical composition suitable for intravenous infusion intoan individual.

The term “pharmaceutically acceptable” as used herein pertains tocompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgement, suitable for use in contactwith the tissues of a subject (e.g., human) without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. Each carrier,excipient, etc. must also be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation.

An aspect of the invention provides a population of modified T cellsexpressing a recombinant cAMP PDE or fragment thereof and an antigenreceptor which binds specifically to cancer cells. A suitable populationmay be produced by a method described above.

The population of modified T cells may be for use as a medicament. Forexample, a population of modified T cells as described herein may beused in cancer immunotherapy therapy, for example adoptive T celltherapy.

Other aspects of the invention provide the use of a population ofmodified T cells as described herein for the manufacture of a medicamentfor the treatment of cancer, a population of modified T cells asdescribed herein for the treatment of cancer, and a method of treatmentof cancer may comprise administering a population of modified T cells asdescribed herein to an individual in need thereof.

The population of modified T cells may be autologous i.e. the modified Tcells were originally obtained from the same individual to whom they aresubsequently administered (i.e. the donor and recipient individual arethe same). A suitable population of modified T cells for administrationto the individual may be produced by a method comprising providing aninitial population of T cells obtained from the individual, modifyingthe T cells to express a cAMP PDE or fragment thereof and an antigenreceptor which binds specifically to cancer cells in the individual, andculturing the modified T cells.

The population of modified T cells may be allogeneic i.e. the modified Tcells were originally obtained from a different individual to theindividual to whom they are subsequently administered (i.e. the donorand recipient individual are different). The donor and recipientindividuals may be HLA matched to avoid GVHD and other undesirableimmune effects. A suitable population of modified T cells foradministration to a recipient individual may be produced by a methodcomprising providing an initial population of T cells obtained from adonor individual, modifying the T cells to express a cAMP PDE orfragment thereof and an antigen receptor which binds specifically tocancer cells in the recipient individual, and culturing the modified Tcells.

Following administration of the modified T cells, the recipientindividual may exhibit a T cell mediated immune response against cancercells in the recipient individual. This may have a beneficial effect onthe cancer condition in the individual.

Cancer conditions may be characterised by the abnormal proliferation ofmalignant cancer cells and may include leukaemias, such as AML, CML, ALLand CLL, lymphomas, such as Hodgkin lymphoma, non-Hodgkin lymphoma andmultiple myeloma, and solid cancers such as sarcomas, skin cancer,melanoma, bladder cancer, brain cancer, breast cancer, uterus cancer,ovary cancer, prostate cancer, lung cancer, colorectal cancer, cervicalcancer, liver cancer, head and neck cancer, oesophageal cancer, pancreascancer, renal cancer, adrenal cancer, stomach cancer, testicular cancer,cancer of the gall bladder and biliary tracts, thyroid cancer, thymuscancer, cancer of bone, and cerebral cancer, as well as cancer ofunknown primary (CUP).

Cancer cells within an individual may be immunologically distinct fromnormal somatic cells in the individual (i.e. the cancerous tumour may beimmunogenic). For example, the cancer cells may be capable of elicitinga systemic immune response in the individual against one or moreantigens expressed by the cancer cells. The tumour antigens that elicitthe immune response may be specific to cancer cells or may be shared byone or more normal cells in the individual.

An individual suitable for treatment as described above may be a mammal,such as a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine(e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. ahorse), a primate, simian (e.g. a monkey or ape), a monkey (e.g.marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orang-utan,gibbon), or a human.

In preferred embodiments, the individual is a human. In other preferredembodiments, non-human mammals, especially mammals that areconventionally used as models for demonstrating therapeutic efficacy inhumans (e.g. murine, primate, porcine, canine, or rabbit animals) may beemployed.

In some embodiments, the individual may have minimal residual disease(MRD) after an initial cancer treatment.

An individual with cancer may display at least one identifiable sign,symptom, or laboratory finding that is sufficient to make a diagnosis ofcancer in accordance with clinical standards known in the art. Examplesof such clinical standards can be found in textbooks of medicine such asHarrison's Principles of Internal Medicine, 15th Ed., Fauci A S et al.,eds., McGraw-Hill, New York, 2001. In some instances, a diagnosis of acancer in an individual may include identification of a particular celltype (e.g. a cancer cell) in a sample of a body fluid or tissue obtainedfrom the individual.

Treatment may be any treatment and therapy, whether of a human or ananimal (e.g. in veterinary applications), in which some desiredtherapeutic effect is achieved, for example, the inhibition or delay ofthe progress of the condition, and includes a reduction in the rate ofprogress, a halt in the rate of progress, amelioration of the condition,cure or remission (whether partial or total) of the condition,preventing, delaying, abating or arresting one or more symptoms and/orsigns of the condition or prolonging survival of a subject or patientbeyond that expected in the absence of treatment.

Treatment may also be prophylactic (i.e. prophylaxis). For example, anindividual susceptible to or at risk of the occurrence or re-occurrenceof cancer may be treated as described herein. Such treatment may preventor delay the occurrence or re-occurrence of cancer in the individual.

In particular, treatment may include inhibiting cancer growth, includingcomplete cancer remission, and/or inhibiting cancer metastasis. Cancergrowth generally refers to any one of a number of indices that indicatechange within the cancer to a more developed form. Thus, indices formeasuring an inhibition of cancer growth include a decrease in cancercell survival, a decrease in tumour volume or morphology (for example,as determined using computed tomographic (CT), sonography, or otherimaging method), a delayed tumour growth, a destruction of tumourvasculature, improved performance in delayed hypersensitivity skin test,an increase in the activity of T cells, and a decrease in levels oftumour-specific antigens. Administration of T cells modified asdescribed herein may improve the capacity of the individual to resistcancer growth, in particular growth of a cancer already present thesubject and/or decrease the propensity for cancer growth in theindividual.

The modified T cells or the pharmaceutical composition comprising themodified T cells may be administered to a subject by any convenientroute of administration, whether systemically/peripherally or at thesite of desired action, including but not limited to; parenteral, forexample, by infusion. Infusion involves the administration of the Tcells in a suitable composition through a needle or catheter. Typically,T cells are infused intravenously or subcutaneously, although the Tcells may be infused via other non-oral routes, such as intramuscularinjections and epidural routes. Suitable infusion techniques are knownin the art and commonly used in therapy (see, e.g., Rosenberg et al.,New Eng. J. of Med., 319:1676, 1988).

Typically, the number of cells administered is from about 10⁵ to about10¹⁰ per Kg body weight, typically 2×10⁸ to 2×10¹⁰ cells per individual,typically over the course of 30 minutes, with treatment repeated asnecessary, for example at intervals of days to weeks. It will beappreciated that appropriate dosages of the modified T cells, andcompositions comprising the modified T cells, can vary from patient topatient. Determining the optimal dosage will generally involve thebalancing of the level of therapeutic benefit against any risk ordeleterious side effects of the treatments of the present invention. Theselected dosage level will depend on a variety of factors including, butnot limited to, the activity of the particular cells, the route ofadministration, the time of administration, the rate of loss orinactivation of the cells, the duration of the treatment, other drugs,compounds, and/or materials used in combination, and the age, sex,weight, condition, general health, and prior medical history of thepatient. The amount of cells and the route of administration willultimately be at the discretion of the physician, although generally thedosage will be to achieve local concentrations at the site of actionwhich achieve the desired effect without causing substantial harmful ordeleterious side-effects.

While the modified T cells may be administered alone, in somecircumstances the modified T cells may be administered cells incombination with the target antigen, APCs displaying the target antigen,and/or IL-2 to promote expansion in vivo of the population of modified Tcells. The population of modified T cells may be administered incombination with one or more other therapies, such as cytokines e.g.IL-2, cytotoxic chemotherapy, radiation and immuno-oncology agents,including checkpoint inhibitors, such as anti-B7-H3, anti-B7-H4,anti-TIM3, anti-KIR, anti-LAG3, anti-PD-1, anti-PD-L1, and anti-CTLA4antibodies.

The one or more other therapies may be administered by any convenientmeans, preferably at a site which is separate from the site ofadministration of the modified T cells.

Administration of modified T cells can be effected in one dose,continuously or intermittently (e.g., in divided doses at appropriateintervals) throughout the course of treatment. Methods of determiningthe most effective means and dosage of administration are well known tothose of skill in the art and will vary with the formulation used fortherapy, the purpose of the therapy, the target cell being treated, andthe subject being treated. Single or multiple administrations can becarried out with the dose level and pattern being selected by thetreating physician. Preferably, the modified T cells are administered ina single transfusion of a least 1×10⁹ T cells.

Other aspects of the invention provide nucleic acids and other reagentsfor the generation of modified T cells as described herein.

An isolated nucleic acid may comprise a nucleotide sequence encoding anantigen receptor which binds specifically to cancer cells and anucleotide sequence encoding a cAMP PDE or a fragment of a cAMP PDE.

The coding sequences may be operably linked to the same or differentpromoters or other regulatory elements. Suitable promoters are wellknown in the art and include mammalian promoters, such as Humanelongation factor-1 alpha (EF1α). In some embodiments, the codingsequences may be separated by a cleavage recognition sequence. Thisallows the cAMP PDE or fragment and antigen receptor to be expressed asa single fusion which undergoes intracellular cleavage by a sitespecific protease, such as furin, to generate the two separate proteins.Suitable cleavage recognition sequences include 2A-furin sequence.Examples of single fusions include SEQ ID NO: 6, which comprises aMAGE-A4 TCR and an N terminal PDE7A fragment separated by a furincleavage site; and SEQ ID NO: 8, which comprises a NY-ESO TCR and an Nterminal PDE7A fragment separated by a furin cleavage site.

Examples of suitable isolated nucleic acids include SEQ ID NO: 5, whichencodes a fusion comprising a MAGE-A4 TCR and an N terminal PDE7Afragment separated by a furin cleavage sequence; and SEQ ID NO: 7, whichencodes a fusion comprising an NY-ESO TCR and an N terminal PDE7Afragment separated by a furin cleavage site.

The nucleotide sequences encoding the antigen receptor and the cAMP PDEor fragment may be located in the same expression vector. For example, asuitable expression vector may comprise a nucleic acid as describedabove. Alternatively, the coding sequences may be located in separateexpression vectors.

Suitable vectors are well known in the art and are described in moredetail above.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorfragments, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Preferably, the vector containsappropriate regulatory sequences to drive the expression of the nucleicacid in mammalian cells. A vector may also comprise sequences, such asorigins of replication, promoter regions and selectable markers, whichallow for its selection, expression and replication in bacterial hostssuch as E. coli. Preferred vectors include retroviral vectors, such aslentiviral vectors, including VSVg-pseudotyped self-inactivatingvectors.

A viral vector, such as a lentivirus, may be contained in a viralparticle comprising the nucleic acid vector encapsulated by one or moreviral proteins. A viral particle may be produced by a method comprisingtransducing mammalian cells with a viral vector as described herein andone or more viral packaging and envelope vectors and culturing thetransduced cells in a culture medium, such that the cells producelentiviral particles that are released into the medium.

Following release of viral particles, the culture medium comprising theviral particles may be collected and, optionally the viral particles maybe concentrated.

Following production and optional concentration, the viral particles maybe stored, for example by freezing at −80° C. ready for use intransducing T cells.

Other aspects and embodiments of the invention provide the aspects andembodiments described above with the term “comprising” replaced by theterm “consisting of” and the aspects and embodiments described abovewith the term “comprising” replaced by the term “consisting essentiallyof”.

It is to be understood that the application discloses all combinationsof any of the above aspects and embodiments described above with eachother, unless the context demands otherwise. Similarly, the applicationdiscloses all combinations of the preferred and/or optional featureseither singly or together with any of the other aspects, unless thecontext demands otherwise.

Modifications of the above embodiments, further embodiments andmodifications thereof will be apparent to the skilled person on readingthis disclosure, and as such, these are within the scope of the presentinvention.

All documents and sequence database entries mentioned in thisspecification are incorporated herein by reference in their entirety forall purposes.

“and/or” where used herein is to be taken as specific disclosure of eachof the two specified features or components with or without the other.For example “A and/or B” is to be taken as specific disclosure of eachof (i) A, (ii) B and (iii) A and B, just as if each is set outindividually herein.

Certain aspects and embodiments of the invention will now be illustratedby way of example and with reference to the figures described above.

Experiments

1. Materials and Methods

1.1 Lentiviral Vectors and Viral Particle Production

The lentiviral system was a 3^(rd) generation VSVg-pseudotypedself-inactivating vector used to express phosphodiesterase (PDE) 4C orPDE7A containing an N-terminal human Influenza hemagglutinin tag orPDE7A or a fragment of PDE7A all in tandem with the NY-ESO1^(c259) TCRα- and β-chains from the EF 1 α promoter. Each transgene was separatedby a 2A-furin sequence.

The plasmid pELNS-ADB1076 comprises a PDE4C (FIG. 1a ) or PDE7A (FIG. 1b) upstream of the TCR. In addition to the EF-1a promoter and downstreamPDE and TCR transgenes, various elements representing lentivirus andbacterial plasmid features are shown. Maps were generated with the CLCMain Workbench software version 7.6.1.

Lentiviral particles were produced by transfecting HEK 293T cells with a3 plasmid system encoding the packaging and envelope elements as well asthe lentivector plasmid using the Turbofect transfection reagent(ThermoScientific). Supernatants were collected 48-72 hours aftertransfection, centrifuged and filtered prior to overnight concentrationat 10,000×g. Medium was removed so that lentiviral particles wereconcentrated 5-10-fold compared with the unconcentrated supernatants.Lentivirus preparations were then snap-frozen on dry ice and stored at−80° C. until used.

1.2 PBMC Isolation and T Cell Expansion

PBMCs isolated from the venous blood of healthy human volunteers weresubjected to CD14+ cell depletion, incubated overnight with CD3/CD28antibody coated beads overnight in IMDM medium (Gibco) supplemented with10% foetal bovine serum (FBS), 1% penicillin and streptomycin and 1%L-glutamine in the presence of IL-2, followed by lentiviral transductionto express either the enhanced affinity NY-ESO1^(c259) TCR or theNY-ESO1^(c259) TCR expressed in tandem with HA-PDE7A, HA-PDE4C, PDE7A ora fragment of PDE7A. T cells were then expanded in culture for 14 daysand TCR transduction levels were assessed by flow cytometry by stainingfor Vβ313.1 (TRBV 6-9).

Lentiviral transduction was performed using the 4 vector systemdescribed in Dull, T., et al (1998). J. Virol. 72, 8463-8471.

1.3 Cancer Cells

NY-ESO1-positive A375 melanoma cells purchased from the ATCC (CRL-1619)were cultured in RPMI 1640 (Gibco) supplemented with 10% FBS, 1%penicillin and streptomycin and 1% L-glutamine (R10). A375 melanomacells were harvested using Trypsin/0.25% EDTA and washed prior to use astargets in T cell activation assays.

1.4 Reagents

Adenosine, prostaglandin E2 (PGE2) and forskolin were purchased fromSigma Aldrich. Adenosine powder was diluted in 0.13 M NH₄OH diluted inPBS to a final concentration of 25 mM. Forskolin was diluted indimethyl-sulfoxide (DMSO) at 100 mM. Prostaglandin E2 was diluted in PBScontaining 10% ethanol at 285 μM. Each reagent was used at the finalconcentrations indicated in section 2.

1.5 IFN-γ ELISA Assay and Cytokine Measurement

A375 melanoma target cells were harvested, resuspended and plated out in96 well flat bottom plates at 50,000 target cells/well. T cells wereeither used fresh following expansion or cryopreserved, thawed andwashed. T cells were rested for approximately 2 hours at 37° C. in R10medium, before plating out at a concentration of 120,000-200,000 Tcells/well.

Adenosine, PGE2 or forskolin were added at the indicated finalconcentration to make the final volume to 200 μl/well. Each assaycondition was performed in triplicates. Cells were cultured for 48 hoursat 37° C. and supernatants were collected before freezing and storing at−20° C. Prior to any assay, the plates were thawed before centrifuging.Supernatants were then transferred to new 96 well plates for subsequentuse in assays.

IFN-γ concentrations were determined using the human DuoSet ELISA kits(R&D Systems) according to the manufacturer's instructions using 96 wellhalf area plates. The assays were developed using commercial TMBsubstrate solution and the reaction stopped with 1M H₂SO₄. Assays wereread using the Spectrostar Omega at 450 nm with wavelength correctionset to 540 nm. Data were analysed using Spectrostar data analysissoftware.

1.6 Time-resolved Cytotoxicity Assays

IncuCyte™ FLR technology (Essen Bioscience) enables direct visualisationof caspase-3/7 dependent apoptosis by microscopy at 37° C. in real time.Kinetic measurement of apoptosis is made using CellPlayer™ 96-WellKinetic Caspase-3/7 reagent (Essen Biosciences) which couples theactivated caspase-3/7 recognition motif (DEVD) to Nuncview™ 488, a DNAintercalating dye. When added to tissue culture medium, this inert andnon-fluorescent substrate crosses the cell membrane where it is cleavedby activated caspase-3/7 resulting in the release of the DNA dye andgreen fluorescent staining of the nuclear DNA. Kinetic activation ofcaspase-3/7 can be monitored morphologically using live cell imaging,and quantified using IncuCyte object counting algorithm as the number offluorescent objects per mm². Apoptotic T cells are gated out theanalysis by size exclusion setting filter threshold at 100 μm².

IncuCyte assays were carried out as follows. Briefly, 24 hours prior toassay set up, A375 melanoma target cells were plated at 15,000-20,000cells per well in 50 μl R10. On the day of the assay, prior to addingthe effector cells, 50 μl per well of assay medium containing 10 μMCellPlayer™ 96-Well Kinetic Caspase-3/7 reagent in the presence orabsence of forskolin or PGE2. Non-transduced and transduced effectorcells were plated at 60,000 cells per well in 50 μl assay medium, intriplicate wells, to give a final volume of 150 μl. The IncuCyte was setto take images of each well using a 10-fold magnification every 3-4hours over 96 hours (4 days) at 37° C/5% CO₂.

2. Results

2.1 Target Cell Killing Kinetics of T Cells Expressing NY-ESO1^(c259)andPDE7A

The ability of T cells expressing NY-ESO1^(c259) TCR or NY-ESO1^(c259)TCR in tandem with HA-PDE7A to induce apoptosis of A375 target cells wasmeasured by time-resolved cytotoxicity assay. 60,000 T cells withdifferent transduction states (Non-transduced (NTDs), c259 TCR (ADB869),c259 TCR+PDE7A (ADB1077)) were added to each assay well and co-incubatedwith HLA-A2+/NYESO+A375 melanoma target cells seeded at 15,000 cells perwell 24 hours prior to the assay. CellPlayer™ 96-Well KineticCaspase-3/7 reagent was added to all wells at a final concentration of3.3 μM. Images were taken at intervals of 4 hours over a duration of 96hours. The number of objects/mm², a measure of target cells undergoingapoptosis, was determined for each image and plotted against time.

T cells transduced with NY-ESO1^(c259) TCR in tandem with HA-PDE7Ashowed an improved basal ability to induce apoptosis in A375 targetcells (FIGS. 2a and 2b ). Data points show mean values and standarderror to the mean of triplicate wells. FIGS. 2a and 2b representexperiments performed with T cells isolated from 2 healthy donors. Datashown are representative of 5 different experiments.

2.2 Target Cell Killing Kinetics of T Cells Expressing NY-ESO1^(c259)andPDE7A in the Presence of PGE2

The ability of T cells expressing NY-ESO1^(c259) TCR or NY-ESO1^(c259)TCR in tandem with HA-PDE7A to induce apoptosis of A375 target cells wasmeasured by time-resolved cytotoxicity assay in the absence and presenceof prostaglandin E2.

60,000 T cells with different transduction status were added to eachassay well and co-incubated with HLA-A2+/NYESO+A375 melanoma targetcells seeded at 15,000 per well 24 hours prior to the assay.Prostaglandin E2 was added at a final concentration of 1 μM in eachwell. CellPlayer™ 96-Well Kinetic Caspase-3/7 reagent was added to allwells at a final concentration of 3.3 μM. Images were taken at intervalsof 4 hours over a duration of 96 hours. The number of objects/mm², ameasure of target cells undergoing apoptosis, was determined for eachimage and plotted against time.

Target A375 cells alone, non-transduced cells (NTDs), T cells expressingNY-ESO1^(c259) TCR (ADB869), and T cells expressing both NY-ESO1^(c259)TCR and PDE7A (ADB1077) were investigated for their ability to induceapoptosis of the target cells in the absence (closed symbols) orpresence (open symbols) of PGE2. The overexpression of PDE7A in NY-ESOspecific T cells confers resistance to the inhibitory effects of PGE2(FIGS. 3a and 3b ). The T cells transduced with PDE7A showed killingkinetics with PGE2 present similar to that shown by PDE7A/NY-ESO1^(c259)TCR T cells in the absence of PGE2.

Data points show mean values and standard error to the mean oftriplicate wells. FIGS. 3a and 3b represent experiments performed with Tcells isolated from 2 healthy donors. Data shown are representative of 6experiments.

2.3 Target Cell Killing Kinetics of T Cells Expressing NY-ESO1^(c259)and PDE7A in the Presence of Forskolin

The ability of T cells expressing NY-ESO1^(c259) TCR or NY-ESO1^(c259)TCR in tandem with HA-PDE7A to induce apoptosis of A375 target cells wasmeasured by time-resolved cytotoxicity assay in the absence and presenceof forskolin.

60,000 T cells with different transduction status, as indicated in thekey, were added to each assay well and co-incubated withHLA-A2+/NYESO+A375 melanoma target cells seeded at 15,000 per well 24hours prior to the assay. Forskolin was added at a final concentrationof 30 μM in each well. CellPlayer™ 96-Well Kinetic Caspase-3/7 reagentwas added to all wells at a final concentration of 3.3 μM. Images weretaken at intervals of 4 hours over a duration of 96 hours. The number ofobjects/mm², a measure of target cells undergoing apoptosis, wasdetermined for each image and plotted against time.

Non-transduced cells (NTDs), T cells expressing NY-ESO1^(c259) TCR(ADB869), and T cells expressing both NY-ESO1^(c259) TCR and PDE7A(ADB1077) were investigated for their ability to induce apoptosis of thetarget cells in the absence (closed symbols) or presence (open symbols)of forskolin. The overexpression of PDE7A in NY-ESO specific T cellsconfers partial resistance to the inhibitory effects of forskolin ontarget cell killing ability (FIGS. 4a and 4b ). Data points show meanvalues and standard error to the mean of triplicate wells. FIGS. 4a and4b represent experiments performed with T cells isolated from 2 healthydonors. Data shown are representative of 6 different experiments.

2.4 IFN-γ Secretion of T Cells Expressing NY-ESO1^(c259) and PDE7A

Forskolin, adenosine and PGE2 can increase intracellular cAMP levels. Toinvestigate the effect of these mediators on IFN-γ secretion from Tcells, cytokine analysis was performed on T cells expressingNY-ESO1^(c259) TCR and T cells expressing both NY-ESO1^(c259) TCR andPDE7A by sandwich ELISA.

200,000 T cells isolated from a healthy donor transduced withlentivectors expressing the NY-ESO1-specific TCR c259 alone or in tandemwith phosphodiesterase 7A (PDE7A) were added to each assay well andco-incubated with HLA-A2+/NYESO+A375 melanoma target cells seeded at50,000 per well. Forskolin was added at a final concentration of 30 μM,adenosine at 250 μM and prostaglandin E2 at 1 or 0.3 μM. T cells andtarget cells were co-cultured for 48 hours and the supernatants werecollected for cytokine analysis.

T cells expressing PDE7A are partially resistant to the inhibition ofIFN-γ release by forskolin (FIG. 5a ), adenosine (FIG. 5b ), and PGE2(FIG. 5c ). Data shown are representative of 6 experiments.

2.5 IFN-γ Secretion of T Cells Expressing NY-ESO1^(c259) and PDE4C

Sandwich ELISA was employed to investigate the effect of forskolin andPGE2 on IFN-γ secretion from T cells expressing NY-ESO1^(c259) TCR and Tcells expressing both NY-ESO1^(c259) TCR and PDE4C.

200,000 T cells isolated from a healthy donor transduced withlentivectors expressing the NY-ESO1-specific TCR c259 alone or in tandemwith phosphodiesterase 4C (PDE4C) were added to each assay well andco-incubated with HLA-A2+/NYESO+A375 melanoma target cells seeded at50,000 per well. Forskolin was added at a final concentration of 30 μMand prostaglandin E2 at 1 μM. T cells and target cells were co-culturedfor 48 hours and the supernatants were collected for cytokine analysis.

T cells expressing PDE4C show partial resistance to the inhibitoryeffects of PGE2 and forskolin on IFN-γ release (FIG. 6A and 6B). FIG. 6Aand 6B represent experiments performed with T cells isolated from 2healthy donors.

2.6 Biological Titre of Lentiviral Constructs Containing NY-ESO1^(c259)TCR and Full-Length or Truncated Forms of PDE7A

The various lentiviral preparations were titrated on PBL to determinetheir effective biological titre. T cell transduction levels weredetermined by staining for the Vβ chain of the TCR (Vβ13.1).

When using the same concentration of lentivirus, T cell transduction wasalways much lower for the NY-ESO1^(c259) TCR+full length PDE7Atransduced cells compared to cells transduced with NY-ESO1^(c259) TCRalone, while the transduction was at an intermediate level forNY-ESO1^(c259) TCR+truncated PDE7A transduced cells (FIG. 7). Thedifferences in transduction efficiency can probably be explained by thedifferent sizes of the constructs.

2.7 Transduction Efficiency and TCR Expression Level of T CellsExpressing NY-ESO1^(c259) and Full-Length or Truncated Forms of PDE7A

T cell transduction levels were determined by staining for the Vβ chainof the TCR (Vβ13.1). The amount of virus used for transduction wasnormalised after lentiviral titration leading to very similartransduction efficiencies for all constructs (FIG. 8A). The TCRexpression level as measured by median fluorescence intensity (MFI) ofVβ13.1 was considerably lower for T cells transduced with NY-ESO1^(c259)TCR+full length PDE7A compared to T cells transduced with NY-ESO1^(c259)TCR alone (FIG. 8B). T cells transduced with NY-ESO1^(c259)TCR+truncated PDE7A showed intermediate TCR expression levels.

2.8 Target Cell Killing Kinetics of T Cells Expressing NY-ESO1^(c259)andFull-Length or Truncated Forms of PDE7A in the Presence of PGE2

The ability of T cells expressing (i) NY-ESO1^(c259) TCR (ii)NY-ESO1^(c259) TCR and full-length PDE7A or (iii) NY-ESO1^(c259) TCR andtruncated PDE7A to induce apoptosis of A375 target cells was measured bytime-resolved cytotoxicity assay in the absence and presence ofprostaglandin E2.

60,000 T cells with different transduction status were added to eachassay well and co-incubated with HLA-A2+/NY-ESO+ A375 melanoma targetcells seeded at 20,000 per well 24 hours prior to the assay.Prostaglandin E2 was added at a final concentration of 1 μM in eachwell. CellPlayer™ 96-Well Kinetic Caspase-3/7 reagent was added to allwells at a final concentration of 3.3 μM. Images were taken at intervalsof 3 hours over a duration of 96 hours. The number of objects/mm², ameasure of target cells undergoing apoptosis, was determined for eachimage and plotted against time.

Non-transduced T cells (NTD), T cells expressing NY-ESO1²⁵⁹ TCR, T cellsexpressing both NY-ESO1^(c259) TCR and full-length PDE7A and T cellsexpressing both NY-ESO1^(c259) TCR and truncated PDE7A were investigatedfor their ability to induce apoptosis of the target cells in the absence(closed symbols) or presence (open symbols) of PGE2 (FIG. 9A). Theoverexpression of both full-length and truncated PDE7A in NY-ESOspecific T cells confers resistance to the inhibitory effects of PGE2(FIG. 9c ) although the effect was slightly reduced for the truncatedPDE7A. T cells transduced with both forms of PDE7A showed killingkinetics with PGE2 present that were similar to that shown by thecorresponding cells in the absence of PGE2 (FIG. 9B).

Data points show mean values and standard error to the mean oftriplicate wells. FIG. 9 represents experiments performed with T cellsisolated from one healthy donors. Data shown are representative of 4experiments.

2.9 IFN-γ Secretion of T Cells Expressing NY-ESO1^(c259) and Full-Lengthor Truncated Forms of PDE7A.

Sandwich ELISA was employed to investigate the effect of forskolin andPGE2 on IFN-γ secretion from T cells expressing NY-ESO1^(c259) TCR, Tcells expressing both NY-ESO1^(c259) TCR and full-length PDE7A, and Tcells expressing both NY-ESO1^(c259) TCR and truncated PDE7A.

120,000 T cells isolated from a healthy donor transduced withlentivectors expressing the NY-ESO1-specific TCR c259 alone or in tandemwith full-length phosphodiesterase 7A or truncated phosphodiesterase 7Awere added to each assay well and co-incubated with HLA-A2+/NYESO+A375melanoma cells seeded at 50,000 per well. Forskolin was added at a finalconcentration of 50 μM and prostaglandin E2 at 0.3 μM and 1 μM. T cellsand target cells were co-cultured for 48 hours and the supernatants werecollected for cytokine analysis.

T cells expressing both forms of PDE7A showed resistance to theinhibitory effects of PGE2 and forskolin on IFN-γ release (FIG. 10).FIG. 10 represents experiments performed with T cells isolated from 2healthy donors. Data shown are representative of 4 experiments.

SequencesMEVCYQLPVLPLDRPVPQHVLSRRGAISFSSSSALFGCPNPRQLSQRRGAISYDSSDQTALYIRMLGDVRVRSRAGFESERRGSHPYIDFRIFHSQSEIEVSVSARNIRRLLSFQRYLRSSRFFRGTAVSNSLNILDDDYNGQAKCMLEKVGNWNFDIFLFDRLTNGNSLVSLTFHLFSLHGLIEYFHLDMMKLRRFLVMIQEDYHSQNPYHNAVHAADVTQAMHCYLKEPKLANSVTPWDILLSLIAAATHDLDHPGVNQPFLIKTNHYLATLYKNTSVLENHHWRSAVGLLRESGLFSHLPLESRQQMETQIGALILATDISRQNEYLSLFRSHLDRGDLCLEDTRHRHLVLQMALKCADICNPCRTWELSKQWSEKVTEEFFHQGDIEKKYHLGVSPLCDRHTESIANIQIGFMTYLVEPLFTEWARFSNTRLSQTMLGHVGLNKAS WKGLQREQSSSEDTDAAFELNSQLLPQENRLSSEQ ID NO: 1 (PDE7A) NP_001229247.1MEPPTVPSERSLSLSLPGPREGQATLKPPPQHLWRQPRTPIRIQQRGYSDSAERAERERQPHRPIERADAMDTSDRPGLRTTRMSWPSSFHGTGTGSGGAGGGSSRRFEAENGPTPSPGRSPLDSQASPGLVLHAGAATSQRRESFLYRSDSDYDMSPKTMSRNSSVTSEAHAEDLIVTPFAQVLASLRSVRSNFSLLTNVPVPSNKRSPLGGPTPVCKATLSEETCQQLARETLEELDWCLEQLETMQTYRSVSEMASHKFKRMLNRELTHLSEMSRSGNQVSEYISTTFLDKQNEVEIPSPTMKEREKQQAPRPRPSQPPPPPVPHLQPMSQITGLKKLMHSNSLNNSNIPRFGVKTDQEELLAQELENLNKWGLNIFCVSDYAGGRSLTCIMYMIFQERDLLKKFRIPVDTMVTYMLTLEDHYHADVAYHNSLHAADVLQSTHVLLATPALDAVFTDLEILAALFAAAIHDVDHPGVSNQFLINTNSELALMYNDESVLENHHLAVGFKLLQEDNCDIFQNLSKRQRQSLRKMVIDMVLATDMSKHMTLLADLKTMVETKKVTSSGVLLLDNYSDRIQVLRNMVHCADLSNPTKPLELYRQWTDRIMAEFFQQGDRERERGMEISPMCDKHTASVEKSQVGFIDYIVHPLWETWADLVHPDAQEILDTLEDNRDWYYSAIRQSPSPPPEEESRGPGHPPLPDKFQFELTLEEEEEEEISMAQIPCTAQEALTAQGLSGVEEALDATIAWEASPAQESLEVMAQEASLEAELEAVYLTQQAQSTGSAPVAPDEFSSREEFVVAVSHSSPSALALQSPLLPAWRTLSVSEHAPGLPGLPSTAAEVEAQREHQAAKRACSACAGTFGEDTSALPAPGGGGSGGDPT SEQ ID NO: 2 (PDE4A) NP_001104777.1MENLGVGEGAEACSRLSRSRGRHSMTRAPKHLWRQPRRPIRIQQRFYSDPDKSAGCRERDLSPRPELRKSRLSWPVSSCRRFDLENGLSCGRRALDPQSSPGLGRIMQAPVPHSQRRESFLYRSDSDYELSPKAMSRNSSVASDLHGEDMIVTPFAQVLASLRTVRSNVAALARQQCLGAAKQGPVGNPSSSNQLPPAEDTGQKLALETLDELDWCLDQLETLQTRHSVGEMASNKFKRILNRELTHLSETSRSGNQVSEYISRTFLDQQTEVELPKVTAEEAPQPMSRISGLHGLCHSASLSSATVPRFGVQTDQEEQLAKELEDTNKWGLDVFKVAELSGNRPLTAIIFSIFQERDLLKTFQIPADTLATYLLMLEGHYHANVAYHNSLHAADVAQSTHVLLATPALEAVFTDLEILAALFASAIHDVDHPGVSNQFLINTNSELALMYNDASVLENHHLAVGFKLLQAENCDIFQNLSAKQRLSLRRMVIDMVLATDMSKHMNLLADLKTMVETKKVTSLGVLLLDNYSDRIQVLQNLVHCADLSNPTKPLPLYRQWTDRIMAEFFQQGDRERESGLDISPMCDKHTASVEKSQVGFIDYIAHPLWETWADLVHPDAQDLLDTLEDNREWYQSKIPRSPSDLTNPERDGPDRFQFELTLEEAEEEDEEEEEEGEETALAKEALELPDTELLSPEAGPDPGDLPLDNQRTSEQ ID NO: 3 (PDE4C) NP_000914.2MEVCYQLPVLPLDRPVPQHVLSRRGAISFSSSSALFGCPNPRQLSQRRGAISYDSSDQTALYIRMLGDVRVRSRAGFESERRGSHPYIDFRIFHSQSEIEVSVSARNIRRLLSFQRYLRSSRFFRGTAVSNSLNILDDDYNGQAKSEQ ID NO: 4 non-catalytic N terminal fragment of PDE7A (repeats areunderlined and PKA pseudo subtrate sites are in bold)ATGGAAGTGTGCTACCAGCTGCCCGTGCTGCCCCTGGATAGACCTGTGCCTCAGCATGTGCTGAGCAGAAGAGGCGCCATCAGCTTCAGCAGCAGCTCCGCCCTGTTCGGCTGCCCCAATCCTAGACAGCTGAGCCAGAGAAGGGGAGCCATCTCCTACGACAGCAGCGACCAGACCGCCCTGTACATCAGAATGCTGGGCGACGTGCGCGTGCGGAGCAGAGCCGGATTTGAGAGCGAGAGAAGAGGCTCCCACCCCTACATCGACTTCCGGATCTTCCACAGCCAGAGCGAGATCGAGGTGTCCGTGTCCGCCCGGAACATCAGACGGCTGCTGAGCTTCCAGAGATACCTGAGAAGCAGCCGGTTCTTCCGGGGCACCGCCGTGTCCAACAGCCTGAACATCCTGGACGACGACTACAACGGCCAGGCCAAGCGGGCCAAGAGATCTGGATCTGGCGCGCCCGTGAAGCAGACCCTGAACTTTGACCTGCTGAAACTGGCCGGCGACGTGGAAAGCAACCCTGGCCCCATGAAGAAGCACCTGACCACCTTTCTCGTGATCCTGTGGCTGTACTTCTACCGGGGCAACGGCAAGAACCAGGTGGAACAGAGCCCCCAGAGCCTGATCATCCTGGAAGGCAAGAACTGCACTCTGCAGTGCAACTACACCGTGTCCCCCTTCAGCAACCTGCGCTGGTACAAGCAGGATACCGGCAGAGGCCCTGTGTCCCTGACCATCCTGACCTTCAGCGAGAACACCAAGAGCAACGGCCGGTACACCGCCACCCTGGACGCCGATACAAAGCAGAGCAGCCTGCACATCACCGCCTCCCAGCTGAGCGATAGCGCCAGCTACATCTGCGTGGTGTCCGGCGGCACAGACAGCTGGGGCAAGCTGCAGTTTGGCGCCGGAACACAGGTGGTCGTGACCCCCGACATCCAGAACCCTGACCCTGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACTCCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGACAAGACCGTGCTGGATATGCGGAGCATGGACTTCAAGAGCAATAGCGCCGTGGCCTGGTCTAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAACTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAATTTCCAGAACCTGAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAGGTGGCCGGATTCAACCTGCTGATGACCCTGCGGCTGTGGTCCTCTGGCTCTCGGGCCAAGAGAAGCGGCAGCGGCGCCACCAATTTCAGCCTGCTGAAGCAGGCAGGGGATGTGGAAGAGAATCCCGGCCCTAGAATGGCCTCCCTGCTGTTTTTCTGCGGCGCCTTCTACCTGCTGGGGACCGGCAGCATGGACGCTGACGTGACCCAGACCCCCCGGAACAGAATCACCAAGACCGGCAAGCGGATCATGCTGGAATGCAGCCAGACAAAGGGCCACGACCGGATGTACTGGTACAGACAGGATCCAGGACTGGGCCTGAGGCTGATCTACTACAGCTTCGATGTGAAGGACATCAACAAGGGCGAGATCAGCGACGGCTACAGCGTGTCCAGACAGGCCCAGGCCAAGTTCTCCCTGAGCCTGGAAAGCGCCATCCCCAACCAGACCGCCCTGTACTTTTGTGCCACAAGCGGCCAGGGCGCCTACGAGGAACAGTTCTTTGGCCCTGGCACCCGGCTGACAGTGCTGGAAGATCTGAAGAACGTGTTCCCCCCAGAGGTGGCAGTGTTCGAGCCTAGCGAGGCCGAGATCTCCCACACCCAGAAAGCCACACTCGTGTGTCTGGCCACCGGATTCTACCCCGACCATGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTGTCCACCGATCCCCAGCCTCTGAAAGAACAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCAGAAATCACTTCAGATGCCAGGTGCAGTTTTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGATAGGGCCAAGCCCGTGACTCAGATCGTGTCTGCCGAAGCCTGGGGCAGAGCCGATTGCGGCTTTACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTACGCCGTGCTGGTGTCTGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGCTGASEQ ID NO: 5 coding sequence for truncated PDE7A with MAGE-A4 TCRMEVCYQLPVLPLDRPVPQHVLSRRGAISFSSSSALFGCPNPRQLSQRRGAISYDSSDQTALYIRMLGDVRVRSRAGFESERRGSHPYIDFRIFHSQSEIEVSVSARNIRRLLSFQRYLRSSRFFRGTAVSNSLNILDDDYNGQAKRAKRSGSGAPVKQTLNFDLLKLAGDVESNPGPMKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTILTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQFGAGTQVVVTPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSGSRAKRSGSGATNFSLLKQAGDVEENPGPRMASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSGQGAYEEQFFGPGTRLTVLEDLKNVFPREVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG*SEQ ID NO: 6 amino acid sequence for truncated PDE7A (underlined) withMAGE-A4 TCRATGGAAGTGTGCTACCAGCTGCCCGTGCTGCCCCTGGATAGACCTGTGCCTCAGCATGTGCTGAGCAGAAGAGGCGCCATCAGCTTCAGCAGCAGCTCCGCCCTGTTCGGCTGCCCCAATCCTAGACAGCTGAGCCAGAGAAGGGGAGCCATCTCCTACGACAGCAGCGACCAGACCGCCCTGTACATCAGAATGCTGGGCGACGTGCGCGTGCGGAGCAGAGCCGGATTTGAGAGCGAGAGAAGAGGCTCCCACCCCTACATCGACTTCCGGATCTTCCACAGCCAGAGCGAGATCGAGGTGTCCGTGTCCGCCCGGAACATCAGACGGCTGCTGAGCTTCCAGAGATACCTGAGAAGCAGCCGGTTCTTCCGGGGCACCGCCGTGTCCAACAGCCTGAACATCCTGGACGACGACTACAACGGCCAGGCCAAGCGGGCCAAGAGATCTGGAAGCGGAGCCCCTGTGAAGCAGACCCTGAACTTCGATCTGCTGAAACTGGCCGGCGACGTGGAAAGCAACCCTGGCCCCATGGAAACACTGCTGGGACTGCTGATCCTGTGGCTGCAGCTGCAGTGGGTGTCCAGCAAGCAGGAGGTGACCCAGATCCCTGCCGCCCTGAGCGTGCCCGAGGGCGAGAACCTGGTGCTGAACTGCAGCTTCACCGACTCCGCCATCTACAACCTGCAGTGGTTCCGGCAGGACCCCGGCAAGGGCCTGACCAGCCTGCTGCTGATCCAGAGCAGCCAGCGGGAGCAGACCAGCGGACGGCTGAACGCCAGCCTGGACAAGAGCAGCGGCCGGAGCACCCTGTACATCGCCGCCAGCCAGCCCGGCGACAGCGCCACCTACCTGTGCGCTGTGCGGCCTCTGTACGGCGGCAGCTACATCCCCACCTTCGGCAGAGGCACCAGCCTGATCGTGCACCCCTACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGTCTGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAATGTGAGCCAGAGCAAGGACAGCGACGTGTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACCTTCTTCCCCAGCCCCGAGAGCAGCTGCGACGTGAAACTGGTGGAGAAGAGCTTCGAGACCGACACCAACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCAGAATCCTGCTGCTGAAGGTGGCCGGATTCAACCTGCTGATGACCCTGCGGCTGTGGAGCAGCGGCTCCCGGGCCAAGAGAAGCGGATCCGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGAGACGTGGAAGAAAACCCTGGCCCTAGGATGAGCATCGGCCTGCTGTGCTGCGCCGCCCTGAGCCTGCTGTGGGCAGGACCCGTGAACGCCGGAGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAAACCGGCCAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTATCGGCAGGACCCCGGCATGGGCCTGCGGCTGATCCACTACTCTGTGGGAGCCGGAATCACCGACCAGGGCGAGGTGCCCAACGGCTACAATGTGAGCCGGAGCACCACCGAGGACTTCCCCCTGCGGCTGCTGAGCGCTGCCCCCAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCTATGTGGGCAACACCGGCGAGCTGTTCTTCGGCGAGGGCTCCAGGCTGACCGTGCTGGAGGACCTGAAGAACGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACACTGGTGTGTCTGGCCACCGGCTTCTACCCCGACCACGTGGAGCTGTCCTGGTGGGTGAACGGCAAGGAGGTGCACAGCGGCGTGTCTACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGACAGCCGGTACTGCCTGTCCTCCAGACTGAGAGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCCGGTGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATTGTGAGCGCCGAGGCCTGGGGCAGGGCCGACTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCTGCCCTGGTGCTGATGGCTATGGTGAAGCGGAAGGACAGCCGGGGCTAASEQ ID NO: 7 coding sequence for truncated PDE7A with NY-ESO TCRMEVCYQLPVLPLDRPVPQHVILSRRGAISFSSSSALFGCPNPRQLSQRRGAISYDSSDQTALYIRMLGDVRVRSRAGFESERRGSHPYIDFRIFHSQSEIEVSVSARNIRRLLSFQRYLRSSRFFRGTAVSNSLNILDDDYNGQAKRAKRSGSGATVKQTLNFDLLKLAGDVESNPGPMETLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLYGGSYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDSKVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSGSRAKRSGSGATNFSLLKQAGDVEENPGPRMSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG*SEQ ID NO: 8 amino acid sequence for truncated PDE7A (underlined) withNY-ESO TCR

1. A method of producing a population of modified T cells comprising;modifying a population of T cells obtained from a donor individual toexpress a cAMP phosphodiesterase (PDE) or a fragment thereof.
 2. Amethod according to claim 1 wherein the T cells are modified byintroducing a nucleic acid encoding the cAMP PDE or fragment into the Tcells.
 3. A method according to claim 2 wherein the nucleic acidencoding the cAMP PDE or fragment is comprised in an expression vector.4. A method according to claim 1 wherein the T cells express an antigenreceptor which binds specifically to cancer cells from the donorindividual.
 5. A method according to claim 4 wherein the antigenreceptor is a T cell receptor (TCR).
 6. A method according to claim 1wherein the method further comprises modifying the population of T cellsto express an antigen receptor which binds specifically to cancer cells.7. A method according to claim 6 wherein the T cells are furthermodified by introducing a nucleic acid encoding the antigen receptorinto the T cells.
 8. A method according to claim 7 wherein the nucleicacid encoding the antigen receptor is comprised in an expression vector.9. A method according to claim 6 wherein the antigen receptor is aheterologous TCR.
 10. A method according to claim 9 wherein the TCRbinds specifically to an MEW displaying a peptide fragment of a tumourantigen expressed by the cancer cells
 11. A method according to claim 10wherein the tumour antigen is NY-ESO-1, MAGE-A4 or MAGE-A10.
 12. Amethod according to claim 6 wherein the antigen receptor is a chimericantigen receptor (CAR).
 13. A method according to claim 12 wherein theCAR binds specifically to tumour antigen expressed by the cancer cells.14. A method according to claim 13 wherein the tumour antigen isNY-ESO-1, MAGE-A4 or MAGE-A10.
 15. A method according to claim 1 furthercomprising administering the modified population of T cells to arecipient individual.
 16. A method according to claim 15 wherein therecipient individual has a cancer condition.
 17. A method according toclaim 16 wherein the cancer condition is characterized by the presenceof one or more cancer cells which bind to said antigen receptor.
 18. Apopulation of modified T cells which express an antigen receptor whichbinds specifically to cancer cells and a cAMP phosphodiesterase (PDE) orfragment thereof, wherein said cells comprise a heterologous nucleicacid encoding the cAMP phosphodiesterase (PDE).
 19. A method of treatingcancer comprising administering to an individual with cancer apopulation of modified T cells which express an antigen receptor whichbinds specifically to cancer cells and a cAMP phosphodiesterase (PDE) orfragment thereof, wherein said cells comprise a heterologous nucleicacid encoding the cAMP phosphodiesterase (PDE).
 20. A method accordingto claim 19 wherein the cancer is characterized by the presence of oneor more cancer cells which bind to said antigen receptor.